We developed a reproducible cytotoxicity assay using the 253J human bladder cancer cell line as target cells, and performed experiments using fractionated effector cells to determine which types of effector cells were capable of lysing 253J cells. 253J cells were susceptible to lysis by Ig-negative, nonadherent, nonphagocytic, Fc receptor-positive cells that have low affinity receptors for sheep erythrocytes. Using the system we tested for the presence of suppressor cells in regional lymph nodes of bladder cancer patients and found suppression in three of four patients tested to date. We also used a heterologous antiserum against 253J cells to arm non-immune effector cells against 253J targets by preincubating them with the anti-253J serum. Arming was specific for 253J cells and could be blocked by aggregated IgG, suggesting that the mechanism of arming involved Fc binding. Cell fractionation studies revealed the armed effector cells were non-adherent and had Fc receptors; while serum fractionation studies show that the arming factor was IgG antibody. We found that the optimal conditions for measuring Concanavalin A (Con A) inducible suppressor cell precursors varied considerably from subject to subject and were influenced by a number of variables. We performed studies that demonstrated the suppression seen in this system was not due merely to differences in the time course of Con A-activated cells and non-activated cells, and we have now developed an assay that is clinically reproducible. Evaluation of our data by analysis of variance by our biostatistician reveals that the variation within the assay, between assays performed on the same day, and between assays performed on different days, is insignificant. While performing these studies, we examined tumor-draining lymph nodes from nine cancer patients and similar nodes from two controls and found statistically significant suppressor cell activity in seven of nine cancer patients tested and zero of two controls tested to date.